H-Ferritin is essential for macrophages' capacity to store or detoxify exogenously added iron.
Mesquita Gonçalo,Silva Tânia,Gomes Ana C,Oliveira Pedro F,Alves Marco G,Fernandes Rui,Almeida Agostinho A,Moreira Ana C,Gomes Maria Salomé
Macrophages are central cells both in the immune response and in iron homeostasis. Iron is both essential and potentially toxic. Therefore, iron acquisition, transport, storage, and release are tightly regulated, by several important proteins. Cytosolic ferritin is an iron storage protein composed of 24 subunits of either the L- or the H-type chains. H-ferritin differs from L-ferritin in the capacity to oxidize Fe to Fe. In this work, we investigated the role played by H-ferritin in the macrophages' ability to respond to immune stimuli and to deal with exogenously added iron. We used mice with a conditional deletion of the H-ferritin gene in the myeloid lineage to obtain bone marrow-derived macrophages. These macrophages had normal viability and gene expression under basal culture conditions. However, when treated with interferon-gamma and lipopolysaccharide they had a lower activation of Nitric Oxide Synthase 2. Furthermore, H-ferritin-deficient macrophages had a higher sensitivity to iron-induced toxicity. This sensitivity was associated with a lower intracellular iron accumulation but a higher production of reactive oxygen species. These data indicate that H-ferritin modulates macrophage response to immune stimuli and that it plays an essential role in protection against iron-induced oxidative stress and cell death.
Inhibition of iron overload-induced apoptosis and necrosis of bone marrow mesenchymal stem cells by melatonin.
Yang Fan,Li Yuan,Yan Gege,Liu Tianyi,Feng Chao,Gong Rui,Yuan Ye,Ding Fengzhi,Zhang Lai,Idiiatullina Elina,Pavlov Valentin,Han Zhenbo,Ma Wenya,Huang Qi,Yu Ying,Bao Zhengyi,Wang Xiuxiu,Hua Bingjie,Du Zhimin,Cai Benzhi,Yang Lei
Iron overload induces severe damage to several vital organs such as the liver, heart and bone, and thus contributes to the dysfunction of these organs. The aim of this study is to investigate whether iron overload causes the apoptosis and necrosis of bone marrow mesenchymal stem cells (BMSCs) and melatonin may prevent its toxicity. Perls' Prussion blue staining showed that exposure to increased concentrations of ferric ammonium citrate (FAC) induced a gradual increase of intracellular iron level in BMSCs. Trypan blue staining demonstrated that FAC decreased the viability of BMSCs in a concentration-dependent manner. Notably, melatonin protected BMSCs against apoptosis and necrosis induced by FAC and it was vertified by Live/Dead, TUNEL and PI/Hoechst stainings. Furthermore, melatonin pretreatment suppressed FAC-induced reactive oxygen species accumulation. Western blot showed that exposure to FAC resulted in the decrease of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bax and Cleaved Caspase-3, and necrosis-related proteins RIP1 and RIP3, which were significantly inhibited by melatonin treatment. At last, melatonin receptor blocker luzindole failed to block the protection of BMSCs apoptosis and necrosis by melatonin. Taken together, melatonin protected BMSCs from iron overload induced apoptosis and necrosis by regulating Bcl-2, Bax, Cleaved Caspase-3, RIP1 and RIP3 pathways.
Bone and maxillofacial abnormalities in thalassemia: a review of the literature.
Di Matteo R,Liuzza F,Manicone P F,Raffaelli L,Berardi D,Perfetti G,Maccauro G
Journal of biological regulators and homeostatic agents
Thalassemia is an inherited blood disorder due to an imbalanced globin chain synthesis leading to anaemia that requires regular blood transfusions and iron-chelating therapy. Of all organ failures secondary to iron deposit, and all the complications, heart failure still represents the first cause of death. Osteopenia and osteoporosis can be considered important causes of morbidity in a population whose lifespan is getting longer, with a strong impact on their quality of life. Authors have reported mainly bone, oral and maxillofacial abnormalities and relative complications, especially in terms of traumatic risk, in patients affected by thalassemia. As examples, this study reports bone modifications in three clinical cases; one of these was also complicated with a femoral fracture, surgically treated with the same criteria of metastatic femoral bone disease. More research on this topic is necessary for the prevention of several complications caused by this disease, and to carefully plan dental or traumatologic operations.
Iron overload adversely effects bone marrow haematogenesis via SIRT-SOD2-mROS in a process ameliorated by curcumin.
Zhou Shujuan,Sun Lan,Qian Shanhu,Ma Yongyong,Ma Ruye,Dong Yuqing,Shi Yifen,Jiang Songfu,Ye Haige,Shen Zhijian,Zhang Shenghui,Shen Jianping,Yu Kang,Wang Siqian
Cellular & molecular biology letters
BACKGROUND:Iron overload, which is common in patients with haematological disorders, is known to have a suppressive effect on haematogenesis. However, the mechanism for this effect is still unclear. The antioxidant curcumin has been reported to protect against iron overload-induced bone marrow damage through an as-yet-unknown mechanism. METHODS:We established iron overload cell and mouse models. Mitochondrial reactive oxygen species (mROS) levels, autophagy levels and the SIRT3/SOD2 pathway were examined in the models and in the bone marrow of patients with iron overload. RESULTS:Iron overload was shown to depress haematogenesis and induce mitochondrion-derived superoxide anion-dependent autophagic cell death. Iron loading decreased SIRT3 protein expression, promoted an increase in SOD2, and led to the elevation of mROS. Overexpression of SIRT3 reversed these effects. Curcumin treatment ameliorated peripheral blood cells generation, enhanced SIRT3 activity, decreased SOD2 acetylation, inhibited mROS production, and suppressed iron loading-induced autophagy. CONCLUSIONS:Our results suggest that curcumin exerts a protective effect on bone marrow by reducing mROS-stimulated autophagic cell death in a manner dependent on the SIRT3/SOD2 pathway.
Chondrocyte ferroptosis contribute to the progression of osteoarthritis.
Yao Xudong,Sun Kai,Yu Shengnan,Luo Jiahui,Guo Jiachao,Lin Jiamin,Wang Genchun,Guo Zhou,Ye Yaping,Guo Fengjing
Journal of orthopaedic translation
Background:Osteoarthritis (OA) is a complex process comprised of mechanical load, inflammation, and metabolic factors. It is still unknown that if chondrocytes undergo ferroptosis during OA and if ferroptosis contribute to the progression of OA. Materials and methods:In our study, we use Interleukin-1 Beta (IL-1β) to simulate inflammation and ferric ammonium citrate (FAC) to simulate the iron overload . Also, we used the surgery-induced destabilized medial meniscus (DMM) mouse model to induce OA . We verify ferroptosis by its definition that defined by the Nomenclature Committee on Cell Death with both and model. Results:We observed that both IL-1β and FAC induced reactive oxygen species (ROS), and lipid ROS accumulation and ferroptosis related protein expression changes in chondrocytes. Ferrostatin-1, a ferroptosis specific inhibitor, attenuated the cytotoxicity, ROS and lipid-ROS accumulation and ferroptosis related protein expression changes induced by IL-1β and FAC and facilitated the activation of Nrf2 antioxidant system. Moreover, erastin, the most classic inducer of ferroptosis, promoted matrix metalloproteinase 13 (MMP13) expression while inhibited type II collagen (collagen II) expression in chondrocytes. At last, we proved that intraarticular injection of ferrostatin-1 rescued the collagen II expression and attenuated the cartilage degradation and OA progression in mice OA model. Conclusions:In summary, our study firstly proved that chondrocytes underwent ferroptosis under inflammation and iron overload condition. Induction of ferroptosis caused increased MMP13 expression and decreased collagen II expression in chondrocytes. Furthermore, inhibition of ferroptosis, by intraarticular injection of ferrostatin-1, in our case, seems to be a novel and promising option for the prevention of OA. The translational potential of this article:The translation potential of this article is that we first indicated that chondrocyte ferroptosis contribute to the progression of osteoarthritis which provides a novel strategy in the prevention of OA.
The detrimental effects of iron on the joint: a comparison between haemochromatosis and haemophilia.
van Vulpen Lize F D,Roosendaal Goris,van Asbeck B Sweder,Mastbergen Simon C,Lafeber Floris P J G,Schutgens Roger E G
Journal of clinical pathology
Joint damage due to (recurrent) joint bleeding in haemophilia causes major morbidity. Although the exact pathogenesis has not been fully elucidated, a central role for iron is hypothesised. Likewise, in hereditary haemochromatosis joint destruction is caused by iron overload. A comparison between these types of arthropathy could provide more insight in the influence of iron in inducing joint damage. A literature review was performed to compare both disorders with respect to their clinical and histological characteristics, and preclinical studies on the influence of iron on different joint components were reviewed. Similarities in the features of arthropathy in haemochromatosis and haemophilia are cartilage degeneration, subchondral bone changes with osteophyte and cyst formation, and osteoporosis. In both disorders synovial inflammation and proliferation are seen, although this is much more explicit in haemophilia. Other substantial differences are the age at onset, the occurrence of chondrocalcinosis radiographically and calcium pyrophosphate dihydrate deposition disease in haemochromatosis, and a rapid progression with joint deformity and neovascularisation in haemophilia. Preclinical studies demonstrate detrimental effects of iron to all components of the joint, resulting in synovial inflammation and hyperplasia, chondrocyte death, and impaired osteoblast function. These effects, particularly the synovial changes, are aggravated in the presence of a pro-inflammatory signal, which is prominent in haemophilic arthropathy and minimal in haemochromatosis. Additional research is needed to further specify the role of iron as a specific target in treating these types of arthropathy.
Iron overload induced death of osteoblasts in vitro: involvement of the mitochondrial apoptotic pathway.
Tian Qing,Wu Shilei,Dai Zhipeng,Yang Jingjing,Zheng Jin,Zheng Qixin,Liu Yong
BACKGROUND:Iron overload is recognized as a new pathogenfor osteoporosis. Various studies demonstrated that iron overload could induce apoptosis in osteoblasts and osteoporosis in vivo. However, the exact molecular mechanisms involved in the iron overload-mediated induction of apoptosis in osteoblasts has not been explored. PURPOSE:In this study, we attempted to determine whether the mitochondrial apoptotic pathway is involved in iron-induced osteoblastic cell death and to investigate the beneficial effect of N-acetyl-cysteine (NAC) in iron-induced cytotoxicity. METHODS:The MC3T3-E1 osteoblastic cell line was treated with various concentrations of ferric ion in the absence or presence of NAC, and intracellular iron, cell viability, reactive oxygen species, functionand morphology changes of mitochondria and mitochondrial apoptosis related key indicators were detected by commercial kits. In addition, to further explain potential mechanisms underlying iron overload-related osteoporosis, we also assessed cell viability, apoptosis, and osteogenic differentiation potential in bone marrow-derived mesenchymal stemcells(MSCs) by commercial kits. RESULTS:Ferric ion demonstrated concentration-dependent cytotoxic effects on osteoblasts. After incubation with iron, an elevation of intracelluar labile iron levels and a concomitant over-generation of reactive oxygen species (ROS) were detected by flow cytometry in osteoblasts. Nox4 (NADPH oxidase 4), an important ROS producer, was also evaluated by western blot. Apoptosis, which was evaluated by Annexin V/propidium iodide staining, Hoechst 33258 staining, and the activation of caspase-3, was detected after exposure to iron. Iron contributed to the permeabilizatio of mitochondria, leading to the release of cytochrome C (cyto C), which, in turn, induced mitochondrial apoptosis in osteoblasts via activation of Caspase-3, up-regulation of Bax, and down-regulation of Bcl-2. NAC could reverse iron-mediated mitochondrial dysfunction and blocked the apoptotic events through inhibit the generation of ROS. In addition, iron could significantly promote apoptosis and suppress osteogenic differentiation and mineralization in bone marrow-derived MSCs. CONCLUSIONS:These findings firstly demonstrate that the mitochondrial apoptotic pathway involved in iron-induced osteoblast apoptosis. NAC could relieved the oxidative stress and shielded osteoblasts from apoptosis casused by iron-overload. We also reveal that iron overload in bone marrow-derived MSCs results in increased apoptosis and the impairment of osteogenesis and mineralization.
Ferrous and ferric differentially deteriorate proliferation and differentiation of osteoblast-like UMR-106 cells.
Lertsuwan Kornkamon,Nammultriputtar Ketsaraporn,Nanthawuttiphan Supanan,Phoaubon Supathra,Lertsuwan Jomnarong,Thongbunchoo Jirawan,Wongdee Kannikar,Charoenphandhu Narattaphol
Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine
The association between iron overload and osteoporosis has been found in many diseases, such as hemochromatosis, β-thalassemia and sickle cell anemia with multiple blood transfusion. One of the contributing factors is iron toxicity to osteoblasts. Some studies showed the negative effects of iron on osteoblasts; however, the effects of two biological available iron species, i.e., ferric and ferrous, on osteoblasts are elusive. Since most intracellular ionized iron is ferric, osteoblasts was hypothesized to be more responsive to ferric iron. Herein, ferric ammonium citrate (FAC) and ferrous ammonium sulfate (FAS) were used as ferric and ferrous donors. Our results showed that both iron species suppressed cell survival and proliferation. Both also induced osteoblast cell death consistent with the higher levels of cleaved caspase 3 and caspase 7 in osteoblasts, indicating that iron induced osteoblast apoptosis. Iron treatments led to the elevated intracellular iron in osteoblasts as determined by atomic absorption spectrophotometry, thereby leading to a decreased expression of genes for cellular iron import and increased expression of genes for cellular iron export. Effects of FAC and FAS on osteoblast differentiation were determined by the activity of alkaline phosphatase (ALP). The lower ALP activity from osteoblast with iron exposure was found. In addition, ferric and ferrous differentially induced osteoblastic and osteoblast-derived osteoclastogenic gene expression alterations in osteoblast. Even though both iron species had similar effects on osteoblast cell survival and differentiation, the overall effects were markedly stronger in FAC-treated groups, suggesting that osteoblasts were more sensitive to ferric than ferrous.
Differential effects of Fe2+ and Fe3+ on osteoblasts and the effects of 1,25(OH)2D3, deferiprone and extracellular calcium on osteoblast viability under iron-overloaded conditions.
Lertsuwan Kornkamon,Nammultriputtar Ketsaraporn,Nanthawuttiphan Supanan,Tannop Natnicha,Teerapornpuntakit Jarinthorn,Thongbunchoo Jirawan,Charoenphandhu Narattaphol
One of the potential contributing factors for iron overload-induced osteoporosis is the iron toxicity on bone forming cells, osteoblasts. In this study, the comparative effects of Fe3+ and Fe2+ on osteoblast differentiation and mineralization were studied in UMR-106 osteoblast cells by using ferric ammonium citrate and ferrous ammonium sulfate as Fe3+ and Fe2+ donors, respectively. Effects of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and iron chelator deferiprone on iron uptake ability of osteoblasts were examined, and the potential protective ability of 1,25(OH)2D3, deferiprone and extracellular calcium treatment in osteoblast cell survival under iron overload was also elucidated. The differential effects of Fe3+ and Fe2+ on reactive oxygen species (ROS) production in osteoblasts were also compared. Our results showed that both iron species suppressed alkaline phosphatase gene expression and mineralization with the stronger effects from Fe3+ than Fe2+. 1,25(OH)2D3 significantly increased the intracellular iron but minimally affected osteoblast cell survival under iron overload. Deferiprone markedly decreased intracellular iron in osteoblasts, but it could not recover iron-induced osteoblast cell death. Interestingly, extracellular calcium was able to rescue osteoblasts from iron-induced osteoblast cell death. Additionally, both iron species could induce ROS production and G0/G1 cell cycle arrest in osteoblasts with the stronger effects from Fe3+. In conclusions, Fe3+ and Fe2+ differentially compromised the osteoblast functions and viability, which can be alleviated by an increase in extracellular ionized calcium, but not 1,25(OH)2D3 or iron chelator deferiprone. This study has provided the invaluable information for therapeutic design targeting specific iron specie(s) in iron overload-induced osteoporosis. Moreover, an increase in extracellular calcium could be beneficial for this group of patients.
Minocycline ameliorates osteoporosis induced by ovariectomy (OVX) and iron accumulation via iron chelation, bone metabolism regulation and inhibition of oxidative stress.
Zhang Chenglin,Meng Tong,Dan Zhou,Song Dianwen,Wang Naiguo
QJM : monthly journal of the Association of Physicians
OBJECTIVE:To investigate the effect and mechanism of minocycline on iron accumulation-related postmenopausal osteoporosis. METHODS:The present study established a rat model of ovariectomy (OVX), gave rats ferric ammonium citrate (FAC) and treated them with minocycline, then examined the severity of osteoporosis and iron metabolism in rats. To further explore the mechanism, osteoblasts were treated with FAC and minocycline, then their effects on cell viability, apoptosis, alkaline phosphatase (ALP) activity, bone metabolism proteins, iron metabolism proteins, and oxidative stress in osteoblasts were measured. RESULTS:In the animal study, OVX significantly decreased the serum estradiol level. Both OVX and FAC significantly increased the serum ferritin and tibial iron level, which was significantly decreased by minocycline (P < 0.05). Minocycline significantly increased the ratio of BV/TV, Tb.Th and Tb.N (P < 0.05), and the levels of BALP, BGP and CTX, but decreased the levels of TRAP and ratio of RANKL/OPG (P < 0.05 compared to OVX+FAC group). In the cell study, minocycline significantly decreased the cellular iron accumulation and induced cell death and apoptosis (P < 0.05). Minocycline significantly increased the ALP activity, the expression of Collagen I, Osteocalcin and OPG (P < 0.05). Minocycline significantly decreased the expression of Ferritin and hepcidin, and increased the expression of FPN) (P < 0.05). It also significantly decreased the cellular MD) and protein carbonyl level and RO) intensity, but increased the levels of SO) and GP) (P < 0.05). CONCLUSION:Minocycline ameliorated osteoporosis induced by OVX and iron accumulation. The mechanism may involve iron chelation, regulation of bone and iron metabolism, and inhibition of oxidative stress.
ROS-Mediated Necroptosis Is Involved in Iron Overload-Induced Osteoblastic Cell Death.
Tian Qing,Qin Bo,Gu Yufan,Zhou Lijun,Chen Songfeng,Zhang Song,Zhang Shuhao,Han Qicai,Liu Yong,Wu Xuejian
Oxidative medicine and cellular longevity
Excess iron has been reported to lead to osteoblastic cell damage, which is a crucial pathogenesis of iron overload-related osteoporosis. However, the cytotoxic mechanisms have not been fully documented. In the present study, we focused on whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms. Here, we showed that the cytotoxicity of iron overload in osteoblastic cells was mainly due to necrosis, as evidenced by the Hoechst 33258/PI staining, Annexin-V/PI staining, and transmission electronic microscopy. Furthermore, we revealed that iron overload-induced osteoblastic necrosis might be mediated via the RIPK1/RIPK3/MLKL necroptotic pathway. In addition, we also found that iron overload was able to trigger mitochondrial permeability transition pore (mPTP) opening, which is a critical downstream event in the execution of necroptosis. The key finding of our experiment was that iron overload-induced necroptotic cell death might depend on reactive oxygen species (ROS) generation, as N-acetylcysteine effectively rescued mPTP opening and necroptotic cell death. ROS induced by iron overload promote necroptosis via a positive feedback mechanism, as on the one hand N-acetylcysteine attenuates the upregulation of RIPK1 and RIPK3 and phosphorylation of RIPK1, RIPK3, and MLKL and on the other hand Nec-1, siRIPK1, or siRIPK3 reduced ROS generation. In summary, iron overload induced necroptosis of osteoblastic cells in vitro, which is mediated, at least in part, through the RIPK1/RIPK3/MLKL pathway. We also highlight the critical role of ROS in the regulation of iron overload-induced necroptosis in osteoblastic cells.
Musculoskeletal complications associated with pathological iron toxicity and its molecular mechanisms.
Simão Márcio,Cancela M Leonor
Biochemical Society transactions
Iron is fundamental for several biological functions, but when in excess can lead to the development of toxic events. Some tissues and cells are more susceptible than others, but systemic iron levels can be controlled by treating patients with iron-chelating molecules and phlebotomy. An early diagnostic can be decisive to limit the progression of musculoskeletal complications like osteoarthritis and osteoporosis because of iron toxicity. In iron-related osteoarthritis, aggravation can be associated to a few events that can contribute to joints articular cartilage exposure to high iron concentrations, which can promote articular degeneration with very little chance of tissue regeneration. In contrast, bone metabolism is much more dynamic than cartilage, but progressive iron accumulation and ageing can be decisive factors for bone health. The iron overload associated with hereditary diseases like hemochromatosis, hemophilias, thalassemias and other hereditary anaemias increase the negative impact of iron toxicity in joints and bone, as well as in life quality, even when iron levels can be controlled. The molecular mechanisms by which iron can compromise cartilage and bone have been illusive and only in the last 20 years studies have started to shed some light into the molecular mechanisms associated with iron toxicity. Ferroptosis and the regulation of intracellular iron levels is instrumental in the balance between detoxification and induced cell death. In addition, these complications are accompanied with multiple susceptibility factors that can aggravate iron toxicity and should be identified. Therefore, understanding tissues microenvironment and cell communication is fundamental to contextualize iron toxicity.
Hypoxia inhibits RANKL-induced ferritinophagy and protects osteoclasts from ferroptosis.
Ni Shuo,Yuan Yin,Qian Zhi,Zhong Zeyuan,Lv Tao,Kuang Yanbin,Yu Baoqing
Free radical biology & medicine
Ferroptosis is a new form of regulated cell death. Several studies have demonstrated that ferroptosis was involved in multiple diseases. However, the precise role of ferroptosis in osteoporosis remains unclear. Here, we demonstrated that ferroptosis was involved in osteoclasts over the course of RANKL-induced differentiation, and it was induced by iron-starvation response and ferrintinophagy. Mechanistically, under normoxia but not hypoxia, ferroptosis could be induced due to iron-starvation response (increased transferrin receptor 1, decreased ferritin) followed by RANKL stimulation, and this was attributed to the down-regulation of aconitase activity. We further investigated intracellular iron homeostasis and found that ferritinophagy, a process initiated by FTH-NCOA4 complex autophagosome degradation, was activated followed by RANKL stimulation under normoxia. Interestingly, these processes could not be observed under hypoxia. Moreover, we demonstrated that HIF-1α contributed to the decrease of ferritinophagy and autophagy flux under hypoxia. Additionally, HIF-1α impair autophagy flux via inhibition of autophagosome formation under hypoxia in BMDMs. In vivo study, we indicated that HIF-1α specific inhibitor 2ME2 prevent OVX bone loss. In conclusion, our study comprehensively investigated the role of ferroptosis in osteoclasts in vitro and in vivo, and innovatively suggested that targeting HIF-1α and ferritin thus inducing ferroptosis in osteoclasts could be an alternative in treatment of osteoporosis.